An inflammatory reaction is a complex biochemical and cytological phenomena that are manifested physiologically in tissue by edema, pain and leukocyte infiltration. The most effective drugs for the inflammation are glucocorticoids. Glucocorticoids, conventional anti-inflammatory steroidal drugs, have been proved to exhibit an excellent activity against rheumatoid arthritis and others by inhibiting or preventing various inflammatory reactions which is caused by radioactive, mechanical, chemical, infectious and immunological stimulation.
However, as the steroidal anti-inflammatory drugs are used widely, recently various harmful side effects caused by their abuse have resulted in serious problems. The steroidal anti-inflammatory drugs clinically causes two categories of side effects: the symptoms caused by a sudden break after a long-term administration and those others caused by too much use for a long time. After long-term administration of adrenocortical hormones, abruptly stopping it causes symptoms such as general prostration, fever, myalgia, arthralgia, and decrease of appetite, etc. as an outcome of the renal paresis. It also causes the increased opportunity for bacterial and viral infections, body weight increase, body form change and insomnia. So, there have been several problems for their applications, and thus it is desirable to develop a new anti-inflammatory drug without causing any side effects. To avoid these side effects, we need to develop specific inhibitors at certain inflammatory pathway. PLA2 is the initial step enzyme to generate arachidonic acid from phospholipids that causes that inflammation at several steps later as prostaglandins and leukotrienes. Therefore, blocking of PLA2 activation may be the best way to prevent inflammation. Indeed steroid is a powerful therapeutic approach although precise mechanism is not yet clear. One of the key mechanisms of steroid has been proposed that anti-inflammatory effect is mediated by induction of anti-inflammatory proteins. Glucocorticoid induces many proteins such as lipocortins, inhibitory proteins of phospholipase A2 (PLA2)(Flower, R. J. et al., Nature 278, 456–459, 1979). Numerous studies have revealed that their anti-inflammatory effects are mediated by the induction of lipocortins (Flower, R. J., et al., Adv. Inflamm. Res. 7, 61–69, 1984) and uteroglobins (Miele et al., Endocr. Rev. 8, 474˜490, 1987). Lipocortins (annexins) are a class of proteins that share structural and functional features. In the functional feature, Miele et al. identified a region of sequence similarity between uteroglobin and lipocortin-1. Further they designed several synthetic peptides corresponding to the region of highest similarity between uteroglobin and lipocortin-1: nonapeptides, so called antiflammins (AFs), corresponding to uteroglobin residues 39–47 and lipocortin-1 residues 246–254. Both peptides were shown to be phospholipase A2 (PLA2) inhibitors in vitro and were effective in a classic model of acute inflammation in carrageenan-induced rat footpad edema (Miele et al., Nature 335, 726–730, 1988). However, it is controversial whether or not AFs have any inhibitory effect on PLA2 as well as anti-inflammatory activity in vivo (Hope, W. C., et al. Agents Actions 34, 77–80, 1991; Marki, F., et al. FEBS Lett. 264, 171–175, 1990; Van Binsbergen, J., et al FEBS Lett. 247, 293–297, 1989). The existing antiflammin or PLA2 inhibitor alone was not able to show potent anti-inflammatory effects like dexamethasone.
In the structural feature of lipocortins, most of them behave as extrinsic membrane proteins, which bind reversibly to phopholipid membranes in a manner that depends on calcium ions. Also during the epithelial cell differentiation, lipocortin-1 (annexin-1) becomes incorporated into the comified cell envelope via cross-linking by transglutaminase (TGase) and cannot be extracted by SDS (Moore, K. G., et al Exp. Cell Res. 200, 186–195, 1992; Lee, C. H., et al., FEBS Lett. 477, 268–272, 2000). In the oral epithelium, it was found by sequencing proteins that lipocortin-1 constitutes about 10% of all the barrier envelope proteins. Thus lipocortin-1 itself possibly involves in the barrier formation as a major component containing anti-inflammatory function.
Another way of blocking PLA2 can be inhibition of PLA2 stimulation. Interestingly there is a report that PLA2 was stimulated by TGase (Cordella-Miele, E, Miele, L, & Mukherjee, A. B. J. Biol. Chem. 265, 17180–17188, 1990). The increase of catalytic activity of PLA2 was due to conformational change with intra molecular cross-linking by TGase. We have discovered that the inflammatory cytokines such as IFN- or TNF- could increase the expression of TGase (Kim, S.-Y, Jeong, E.-J., & Steinert, P. M. J. Interferon and Cytokine Res. in press, 2002). Consequently increase of TGase may cause increase of PLA2 activity. Therefore, TGase inhibitors could inhibit inflammation through inhibition of PLA2 stimulation.
As a result of the careful researches by the present inventors, sequence similarity between AFs and TGase substrate domain of elafin, which represents a core tetra peptide KVLD in AFs and DPVK in elafin. Elafin, also known as SKALP (skin-derived antileukoproteinase), is 6 kDa preproform that can be activated to 3 kDa active form by proteolysis, which is specific and potent inhibitor of polymorphonuclear (PMN) cell-derived serine proteinases such as elastase and proteinase-3 (Molhuizen, H. O. F. et al., J. Biol. Chem. 268, 12028–32, 1993). Although KVLD is inactive as a PLA2 inhibitor (Miele et al., Nature 335, 726–730, 1988), which could be active for the TGase inhibitor. The present inventors have also discovered that when an antiflammin contains lysine residues as an acyl acceptor, it competes with TGase substrate, and succeeded in synthesizing novel peptides from antiflammin based on this finding.